Naloxone in the formulation of inactivated COVID-19 vaccine candidate improved cellular and humoral immune responses
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Melika Haghighi,1 Akbar Khorasani ,2 pegah karimi,3 Mehdi Mahdavi ,4,*
1. Recombinant Vaccine Research Center, Tehran University of Medical Sciences, Tehran, Iran
2. 2. Department of FMD vaccine production, Razi Vaccine & Serum Research Institute, Agricultural Research, Education & Extension Organization (AREEO) Karaj, Iran
3. Recombinant Vaccine Research Center, Tehran University of Medical Sciences, Tehran, Iran
4. Recombinant Vaccine Research Center, Tehran University of Medical Sciences, Tehran, Iran
- Introduction: COVID-19 is a novel coronavirus from the same family of SARS-COVID-2 that has presented a major threat to public health worldwide. The major sources of the disease that is spread through respiratory droplets and direct contact are currently wild animal hosts and infected patients. The global spread of SARS-CoV-2 has led to an urgent attempt to develop a vaccine. The potency of a vaccine is highly depends on the nature of adjuvant. Opioids are immune modulating agents that improve the immune responses against vaccines. Naloxone (NLX) is an opioid receptor antagonist approved by the FDA and administered to people with opioid peptide-induced respiratory toxicity. Various studies demonstrated that NLX can be used as an adjuvant for designing microbial vaccines. In this study, inactivated COVID-19 virus formulated in Alum and NLX and then immune responses versus the vaccines were assessed in the experimental mice.
- Methods: A SARS-CoV-2 strain was isolated from pharyngeal sample of a patient and cultivated in Vero cell line for mass production. The virus was inactivated with formalin and after purification quantified with Bradford. Then 4µg of inactivated COVID-19 virus in PBS buffer was mixed with 200µg of Alum hydroxide adjuvant and shaked at 100RPM and allowed that the viral particles to adsorb on the surface of Alum gel. One dose of Alum-formulated vaccine was admixed with 200 µg of NLX (10 mg/kg) as inactivated COVID-19 Alum-NLX10 vaccine and one dose of Alum-formulated vaccine was admixed with 60 µg of NLX (3 mg/kg) as inactivated COVID-19 Alum-NLX3 vaccine. In addition, inactivated COVID-19 virus was admixed with Freund adjuvant (50/50) and homogenized using homogenizer to develop a homogen suspension. Six-to eight-week-old male BALB/c mice (N=50) were purchased from Royan Institute of Iran and assigned into 5 experimental groups (N=10) as below;
Group 1: Inactivated COVID-19-Alum vaccine.
Group 2: Inactivated COVID-19- Alum+ NLX 3mg/kg vaccine.
Group 3: Inactivated COVID-19- Alum+ NLX 10mg/kg vaccine.
Group 4: Inactivated COVID-19- Freund adjuvant vaccine.
Group 5: Mice were immunized with PBS as control group.
Experimental mice were immunized subcutaneously with 4µg of vaccine, two times on days 0 and 14 and two weeks after the final immunization, immunologic parameters were assessed.
- Results: Immunization with COVID-19-Alum-NLX10 shows a significant increase of IFN-γ cytokine versus COVID-19-Alum, COVID-19-Alum-NLX3 and control groups (P< 0.0033).
Immunization with COVID-19-Alum-NLX3 shows a significant decrease of IL-4 cytokine versus COVID-19-Alum group (P< 0.0133). The results of CTL activity based on Gr-B secretion showed that immunization with COVID-19-Alum-NLX10 resulted to a significant increase versus COVID-19-Alum and control groups (P<0.0011).
Results of specific IgG titer in the experimental groups showed that immunization with COVID-19-Alum-NLX3, COVID-19-Alum-NLX10 and COVID-19-Freund resulted to an increase of IgG titer versus COVID-19-Alum and control groups. Results of specific IgG1 and IgG2a response showed that all formulations of COVID-19 vaccine induced both of these isotypes.
- Conclusion: It seems that Naloxone in the vaccine formulation of COVID-19 vaccine improved cellular and humoral immune responses in parallel to previous studies on the other microbial vaccines. The results of the present study are encouraging to pursue the study in a clinical trial of human.
- Keywords: Inactivated COVID-19, Alum, Naloxone, Vaccine formulation, Adjuvant.
